Roles of the 5-HT2C receptor on zebrafish sociality.

Serotonin (5-HT) receptors have been implicated in social behavior in vertebrates. Zebrafish (Danio rerio) have been increasingly being used behavioral neuroscience to study the neurobiological correlates of behavior, including sociality. Nonetheless, the role of 5-HT2C receptors in different social functions were not yet studied in this species. Zebrafish were treated with the agonist MK-212 (2 mg/kg) or the antagonist RS-102221 (2 mg/kg) and tested in the social interaction and social novelty tests, conditional approach test, or mirror-induced aggressive displays. MK-212 increased preference for an unknown conspecific in the social investigation test, but also increased preference for the known conspecific in the social novelty test; RS-102221, on the other hand, decreased preference in the social investigation test but increased preference for the novel conspecific in the social novelty test. MK-212 also decreased predator inspection in the conditional approach test. While RS-102221 decreased time in the display zone in the mirror-induced aggressive display test, it increased display duration. Overall, these results demonstrate the complex role of 5-HT2C receptors in different social contexts in zebrafish, revealing a participation in social plasticity in vertebrates.

Hydrogel encapsulation as a handling and vitrification tool for zebrafish ovarian tissue.

Zebrafish is an important animal model, thousands lines have been developed, thus having a great need for their preservation. However, the cryopreservation of fish oocytes is still limited and needs improvement. The sodium alginate hydrogel, in addition to providing support for the cells, has been shown to be a potential cryoprotectant. Therefore, the aim of this study was to evaluate the sodium alginate hydrogel encapsulation technique efficiency during zebrafish ovarian tissue vitrification. The encapsulation methodology was standardized in the first experiment. In Experiment 2, we evaluated four vitrified groups: standard protocol without encapsulation (VS); encapsulated with cryoprotectants (VS1-A); encapsulated with half the cryoprotectants concentration (VS2-A); encapsulated without cryoprotectants (VA). VS treatment (54.6 ± 12.3%; 23.7 ± 9.9%; 12.6 ± 5.0%) did not differ from the VS1-A and VA showed a lower membrane integrity percentage (1.2 ± 1.4%; 0.3 ± 0.6%; 0.5 ± 1.5%). Mitochondrial activity was significantly greater in non-encapsulated treatment (VS) when compared to the encapsulated treatments. VS1-A and VS obtained the lowest lipid peroxidation (39.4 ± 4.4 and 40.5 ± 3.3 nmol MDA/mg respectively) in which VS was not significantly different from the VS2-A treatment (63.6 ± 3.1 nmol MDA/mg), unlike, VA obtained the highest lipid peroxidation level (124.7 ± 7.9 nmol MDA/mg). The results obtained in this study demonstrate that the sodium alginate hydrogel encapsulation technique did not have a cryoprotective action, but maintained the membrane integrity when used the standard concentration of cryoprotectants. However, halving the cryoprotectant concentration of fragments encapsulated in alginate hydrogel did not cause an increase in lipid peroxidation. In addition, it provided support and prevented the oocytes from loosening from the tissue during the vitrification process, being an interesting alternative for later in vitro maturation.

Gonadal morphology and difference in reproductive development of two isolated populations of Astyanax rivularis (Teleostei, Characidae).

Individuals of the same species may present different reproductive tactics depending on the environment in which they develop and mature. The present study aimed to define the gonadal development phases of males and females of Astyanax rivularis and to carry out a comparative analysis of the reproductive development of specimens captured in two isolated environments of the São Francisco River basin in Serra da Canastra, Brazil (Point 1: low vegetation and river showing calm and crystalline waters with small well formations; Point 2: current waters, and well-established areas of arboreal vegetation). Thus, the gonads of A. rivularis specimens were collected, fixed and processed with techniques for light microscopy. Five maturation phases of the females' reproductive cycle were established: immature, developing, spawning capable, regressing and regenerating. Three maturation phases of the males' reproductive cycle were observed: spawning capable, regressing, and regenerating. There are differences in the phases of gonadal development of A. rivularis between the two sampling points so that, possibly, animals upstream of the waterfall demonstrate a delay in the reproductive cycle in relation to animals downstream.

Preliminary study on testicular germ cell isolation and transplantation in an endangered endemic species Brycon orbignyanus (Characiformes: Characidae).

We aimed to develop a simplified protocol for transplantation of Brycon orbignyanus spermatogonial stem cells (SSCs) into Astyanax altiparanae testes. Brycon orbignyanus testes were enzymatically digested and SSC purified by a discontinuous density gradient. Endogenous spermatogenesis was suppressed in A. altiparanae using busulfan or by incubation at 35 °C water, and SSCs from B. orbignyanus labeled with PKH26 were injected into their testes via the urogenital papilla. Twenty-two hours post-transplantation, labeled spermatogonia were observed in A. altiparanae tubular lumen. After 7 days, spermatogonia proliferated in the epithelium, and 21 days post-transplantation, sperm was observed in the lumen. Of surviving host fish, nearly 67% of those treated with busulfan and 85% of those held in warm water showed labeled cells in host germinal epithelium. The present study standardized, by a simple and accessible method, germ cell transplantation between sexually mature Characiformes fish species. This is the first report of xenogenic SSC transplantation in this fish order.

The influence of increased water temperature on the duration of spermatogenesis in a neotropical fish, Astyanax altiparanae (Characiformes, Characidae).

In view of the established climate change scenario and the consequent changes in global temperature, it is essential to study its effects on animal spermatogenesis. Therefore, the aim of this study was to verify the duration of spermatogenesis at different temperatures. For this purpose, 96 male and adult specimens of Astyanax altiparanae were kept in a closed circulation system with water temperature stabilized at 27 °C and 32 °C. Subsequently, the specimens received pulses of BrdU (bromodeoxyuridine) at a concentration of 100 mg/kg/day for 2 consecutive days, and the samples were collected daily for a period of 15 days. Their testes were removed, fixed, processed in historesin, and sectioned in 3 µm, submitted to hematoxylin/eosin staining and to bromodeoxyuridine immunodetection. Partial results of the optimum temperature experiments allowed the classification of A. altiparanae spermatogenic cells in Aund, Adiff, and type B spermatogonia, spermatocytes, spermatids, and spermatozoa. The duration of spermatogenesis was determined as approximately 6 days for animals at a temperature of 27 °C and 1 day for animals at 32 °C. The elevated temperature was also responsible for increasing cell proliferation, resulting in an increase in the number of spermatocytes, spermatids, spermatozoa, and cell death (cell pyknotic). The duration of spermatogenesis in A. altiparanae was directly affected by the elevated water temperature, causing a reduction in the estimated time of spermatogenesis.

Social Stress Increases Anxiety-Like Behavior Equally in Male and Female Zebrafish.

Zebrafish anxiety-like behavior was assessed in the novel tank test after the formation of dominant-subordinate hierarchies. Ten pairs of animals were subjected to dyadic interactions for 5 days, and compared with control animals. After this period, a clear dominance hierarchy was established across all dyads, irrespective of sex. Social status affected parameters of anxiety-like behavior in the novel tank test, with subordinate males and females displaying more bottom-dwelling, absolute turn angle, and freezing than dominant animals and controls. The results suggest that subordinate male and female zebrafish show higher anxiety-like behavior, which together with previous literature suggests that subordination stress is conserved across vertebrates.

Seminal characterization of the Amazonian fire-eye tetra Moenkhausia oligolepis (Günther, 1864).

The seminal characteristics of Moenkhausia oligolepis are described. Three males were induced with a single dose of carp pituitary. Semen was collected 6 h after induction, and diluted in dibasic sodium phosphate extender solution. For motility analysis, 1 µl of diluted semen was added to 10 µl of distilled water to achieve gamete activation. The average duration of total motility was 76.67 s; while the average sperm motility rate at intervals of 15 s was 95.3, 85.3, 59.6, 31.7, 13.0, 4.6 and 1.2%. To determine sperm concentration in samples, 0.5 µl of semen was diluted with 500 µl of glutaraldehyde. An aliquot of 10 µl of this dilution was utilized for cell counting. An average count of 4.97 × 109 ± 3.46 sperm/ml was obtained. Morphological analyses were performed using eosin-nigrosine dye; 20.33% of the sperm were observed to be dead. Live sperm, comprising the other 79.67%, had an average length of approximately 30 µm, with a head diameter of 4.488 ± 0.7 µm; and a flagella plus mid-piece length of 26.071 ± 12.4 µm. Of those sperm, 69% had a normal morphology, while 31% had primary and secondary abnormalities. The observed abnormality rate did not have a detrimental effect on artificial fertilization potential for the species. The description of the seminal characteristics of a species is one of the most important sets of information required for artificial reproduction of fish in captivity. It also contributes significantly to the total biological knowledge of the studied species.

A model to study orienting responses in zebrafish, and applications towards the emotion-cognition interaction.

Orienting responses (ORs) are whole-organism reflexes that are elicited by innocuous stimuli, and which decrease in magnitude after stimulus repetition. ORs represent relatively simple responses that can be used to study attentional processes, and are modulated by the organism's state, including arousal and activation levels, as well as by emotional processes. Here we describe a simple method to study ORs in zebrafish, a model organism increasingly being used in behavioural neuroscience. After presentation of a static visual stimulus, an OR is elicited, characterized by approaching the stimulus and orienting towards it. After repeated stimulation, OR decreases, suggesting habituation. These responses are qualitatively altered by exposure to a fear-eliciting alarm substance (i.e., derived from the skin of a conspecific), since exposed animals avoid the visual stimulus and orient either away from the stimulus or towards it, but at a distance. The protocol can be used to study orienting responses, as well as the impact of fear and arousal on these reflexes.

Testis structure, spermatogonial niche and Sertoli cell efficiency in Neotropical fish.

Neotropical icthyofauna represents one of the most diverse and extreme ecosystems in the world. Likewise, reproduction showed enormous diversity with different reproductive systems, modes and behavior. On the other hand, information on Neotropical fish species, in particular on male reproductive physiology is restricted to few species. This mini-review aimed to compile the existing information on spermatogenesis of Neotropical teleosts focusing on testis structure, spermatogonial niche and Sertoli cell efficiency. The first topic covers the histological analysis of the testicular structure, showing a conserved testicular pattern in relation to the phylogenetic position: basal species present anastomosing tubular testis (e.g. Astyanax altiparanae, Conorhynchos conirostris, Pimelodus maculatus, Lophiosilurus alexandri, Rhinelepis aspera, among others), while derived teleosts showed lobular testis (e.g. Cichlasoma dimerus, Cichla kelberi, Odontesthes bonariensis, Synbranchus marmoratus and others). Next to testicular structure, existing data showed that type A undifferentiated spermatogonia (Aund) is differentially distributed among the Neotropical species. Aund can be restricted at the blind-end of the germinal compartment (O. bonariensis), or spread along the germinal epithelium (A. altiparanae), or even distributed along the germinal epithelium but concentrated at the blind-end (C. kelberi and C. intermedia). Moreover, recent studies in A. altiparanae have demonstrated that within the germinal compartment, Aund have a preferential distribution in areas neighboring the interstitial compartment - the spermatogonial niche. The proximity with the interstitium suggests that interstitial cells, such as Leydig cells, are important for Aund maintenance in the testis. Finally, this mini-review highlighted Sertoli cell efficiency, showing that a single Sertoli cell can support a higher number of germ cells (80-140 spermatids) in Neotropical species evaluated at the moment (e.g. A. altiparanae, Hoplias malabaricus, Poecilia reticulata, Serrasalmus spilopleura, C. intermedia). Overall, this review provided basic and functional information on spermatogenesis of Neotropical species. More studies in this field are necessary since Neotropical region is considered one of the hotspot regions to discovery new species providing, therefore, new opportunities to investigate spermatogenesis in fish.

Biotechnology applied to fish reproduction: tools for conservation.

This review discusses the new biotechnological tools that are arising and promising for conservation and enhancement of fish production, mainly regarding the endangered and the most economically important species. Two main techniques, in particular, are available to avoid extinction of endangered fish species and to improve the production of commercial species. Germ cell transplantation technology includes a number of approaches that have been studied, such as the transplantation of embryo-to-embryo blastomere, embryo-to-embryo differentiated PGC, larvae to larvae and embryo differentiated PGC, transplantation of spermatogonia from adult to larvae or between adults, and oogonia transplantation. However, the success of germ cell transplantation relies on the prior sterilization of fish, which can be performed at different stages of fish species development by means of several protocols that have been tested in order to achieve the best approach to produce a sterile fish. Among them, fish hybridization and triploidization, germline gene knockdown, hyperthermia, and chemical treatment deserve attention based on important results achieved thus far. This review currently used technologies and knowledge about surrogate technology and fish sterilization, discussing the stronger and the weaker points of each approach.

Triploid or hybrid tetra: Which is the ideal sterile host for surrogate technology?

This work was aimed at developing an effective procedure to obtain sterile ideal host fish in mass scale with no endogenous germ cells in the germinal epithelium, owning permanent stem-cell niches able to be colonized by transplanted germ cells in surrogate technology experiments. Thus, triploids, diploid hybrids, and triploid hybrids were produced. To obtain hybrid offspring, oocytes from a single Astyanax altiparanae female were inseminated by sperm from five males (A. altiparanae, A. fasciatus, A. schubarti, Hyphessobrycon anisitsi, and Oligosarcus pintoi). Triploidization was conducted by inhibition of the second polar body release using heat shock treatment at 40 °C for 2 min. At 9-months of age, the offspring from each crossing was histologically evaluated to access the gonadal status of the fish. Variable morphological characteristics of the gonads were found in the different hybrids offspring: normal gametogenesis, gametogenesis without production of gametes, sterile specimens holding germ cells, and sterile specimens without germ cells, which were considered "ideal hosts". However, only in the hybrid derived from crossing between A. altiparanae and A. fasciatus, 100% of the individuals were completely sterile. Among them 83.3% of the male did not present germ cells inside germinal epithelium, having only somatic cells in the gonad. The other 16.7% also presented spermatogonia inside the niches. Such a methodology allows the production of sterile host in mass scale, opening new insights for application of surrogate technologies.

Stereological analysis of gonads from diploid and triploid fish yellowtail tetra Astyanax altiparanae (Garutti & Britski) in laboratory conditions.

This study aimed to examine the gonadal morphology of diploid and triploid fish through stereological analysis. Triploid individuals were obtained after temperature shock (40°C for 2 min) at 2 min post-fertilization and reared until 175 days post-fertilization (dpf). Intact eggs were used to obtain the diploids. Gonads were collected for histological analysis at 83, 114, 144 and 175 dpf. Diploid females and males presented normal oogenesis and spermatogenesis through all the experimental period. Conversely, stereological analysis revealed that triploid females were sterile and oogonia were the prevalent cell type in the ovaries. Triploid males presented increased amounts of spermatocyte cysts and a large area of lumen when compared with diploids and in addition the amount of spermatozoa was lower than that observed for diploids. However, some triploid males presented spermatogenesis similar to diploids. Therefore, we concluded that triploidization is an interesting alternative to produce sterile individuals in A. altiparanae.

Oogenesis in Laetacara araguaiae (Ottoni and Costa, 2009) (Labriformes: Cichlidae).

We aimed to analyze the oogenesis of adult females of the cichlid fish Laetacara araguaiae. The specimens' gonads were removed and processed for light and transmission electron microscopy. Oogenesis in L. araguaiae showed the following characteristics: a germinal epithelium with three types of oogonia (A-undifferentiated, A-differentiated and B-oogonia), oocytes at meiotic prophase stage and ovarian follicle formation. Oocytes showing primary growth with pre-vitellogenic and cortical alveolus were observed. Similar to data for other cichlids, oocytes in secondary growth or vitellogenesis were characterized by the initial deposition of yolk microgranules. The event that characterizes the maturation stage is nucleolus migration, also called the germinal vesicle, to the oocyte periphery in the direction of the micropyle. The follicular complex undergoes several changes throughout the oocyte stages. To the best of our knowledge this study is the first to describe L. araguaiae oogenesis. Moreover, this study is the first step to better understand the reproductive biology of this species, which shows great potential for use as an ornamental fish.

The effects of temperature and busulfan (Myleran) on the yellowtail tetra Astyanax altiparanae (Pisces, Characiformes) spermatogenesis.

We aimed to standardize a protocol to suppress spermatogenesis in the characiform fish, Astyanax altiparanae, for future use as a host in germ cell transplant research, opening opportunities for a range of studies, such as spermatogenesis analyses and transgenesis because this species presents livestock characteristics to be used as a biological model. The effects of the chemotherapeutic busulfan (formulated as Myleran), which is used as medicine, therefore not as toxic to humans manipulation as analytical grade busulfan (Fluka) used in previous studies, were evaluated at physiological temperature of 28 °C, ideal for growth and reproduction of A altiparanae, and also at increased temperature 35 °C. The temperature groups were divided into three treatment groups: busulfan, DMSO only, and an untreated control. Macroscopic, histologic, stereological, and ultrastructure analysis showed that, at 28 °C, busulfan did not cause depletion of germ cells in A altiparanae. However, at 35 °C, sterilization was observed 3 weeks after the initial application. Similar results were obtained with maintenance of fish at 35 °C for a longer period with no accompanying Myleran treatment. This procedure allows reduction in stress and lower mortality resulting from manipulation during busulfan injection and is also suitable for mass treatment because large numbers of fish can be incubated in warm water.